The invention relates to selected peptides of the HIV gag sequence which bring about inhibition of virus synthesis. On testing 41 sequential peptides for inhibition of HIV replication it was found that the overlapping peptides 4 and 5 with the amino-acid sequence EQU WASRELERFAVNPGLLETSEGCRQ and NPGLLETSEGCROILGQLQPSLQT
from the p17 submembrane protein and the two peptides 28 and 29 with the amino-acid sequences EQU ANPDCKTILKALGPAATLEEMMTA and AATLEEMMTACQGVGGPGHKA
from the p24 core protein inhibit the replication of HIV ( overlapping regions are underlined in each case ) . Peptides of this or similar types which contain at least the overlapping regions are suitable as pharmaceuticals for controlling HIV infections.
The disease AIDS caused by HIV represents a great challenge to scientific research in the development of therapeutically active substances and novel vaccines. Even though research extends over the entire spectrum of possible therapeutic approaches, nevertheless only a very few substances promise the prospect of a novel successful therapy. To date the only substance with anti-HIV activity which is approved on the market is .sup.R Retrovir with the active ingredient zidovudine supplied by Wellcome. This nucleotide analog, azidothymidine (AZT), very effectively inhibits in vitro and in vivo the HIV-specific reverse transcriptase but is not free of disadvantageous properties. To date there are no alternatives to AZT therapy.
Investigations of the viral structure of HIV and the part played by the various virus structural proteins in virus maturation have led to the realization that several approaches for effective inhibition of virus synthesis are offered here, the inhibition of viral protease by chemotherapeutics being only one example which is currently being looked at by various research groups. The fact that the gag sequence is one of the highly conserved regions in the HIV genome suggests that this is a very important protein for virus replication; this suggestion is supported by the small differences in sequences between different HIV isolates (see Tab. 1). The process of virus synthesis is initiated by gag protein synthesis and the required myristoylation of the gag protein. Subsequent assemblage takes place on the lipid membrane of the infected cell. Juxtapositioning of gag proteins leads to a protuberance on the cell membrane and the detachment (budding) of "particles" or immature viruses. The fact: that this process takes place even when only the gag gene is inserted into the cell has been shown by investigations with recombinant gag sequences in vaccinia or baculo vectors (Karacostas et al. (1989) Human immunodeficiency virus-like particles produced by vaccinia virus expression vector. Proc. Natl. Acad. Sci. USA, Vol. 86, 8965-8967; Gheysen et al. (1989) Assembly and release of HIV-1 precursor prgag55 virus-like particles from recombinant baculovirus-infected insect cells. Cell, 59, 103-112). Other publications demonstrate that there are regions important for processing within the gag sequence. Several regions relevant for the processing have been identified by introducing specific mutations within the gag sequence (Gottlinger et al. (1990) Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA, Vol. 86, 5781-5785).
However, there has been no evidence whatever that certain regions of the gag sequence code for peptides which inhibit virus synthesis by HIV.
The experiments on which the invention is based investigated 41 mutually overlapping synthetic peptides of the HIV-1 gag sequence for their inhibiting function on virus synthesis in an in vitro test. The peptides were 24 amino acids long and were synthesized in analogy to the sequence published by Ratner et al. (Complete nucleotide sequence of the AIDS virus, HTLV-III, Nature, 313, 277-284 (1985)).
These peptides were added in various concentrations (200-40 .mu.g/ml) to freshly infected jurkat cells. Infection was carried out with 100-1000 TCID.sub.50. The infection was analyzed by microscopic assessment and investigation of the supernatant for the content of infectious HIV. For this purpose, the cell supernatant was added to non-infected jurkat cells. After incubation for two weeks, this detector cell culture was examined for HIV under the microscope and by reverse transcriptase assay. It was found from this that there are two gag regions which exert an inhibitory effect on virus synthesis. These two regions are each represented by two peptides, the first peptide (peptide 4+5) being located inside p17, and the second (peptide 28+29) being located inside p24.
As is evident from Tab. 4, no p24 is detectable in HIV-infected cells after treatment with these peptides. The supernatant from these cell cultures contains no infectious HIV capable of infecting a control culture. FIGS. 1 and 2 report the reverse transcriptase (RT) activity of the two detector cell cultures. The four selected peptides are able very effectively to inhibit HIV synthesis in concentrations of 200-40 .mu.g/ml. (See examples for details of the demonstration of HIV inhibition).
The effect of different concentrations of infectious virus on the course of the experiment is relatively low and is mainly reflected by the level of measured RT activity (cf. Experiment 1, FIG. 1; Experiment 2, FIG. 2). Nor do different concentrations of added peptides have a significant effect on the inhibition, which suggests that the concentration of added peptides can be even further reduced without influencing the effect. The result of the third experiment is reported in FIG. 3. The reverse transcriptase activities depicted here are as measured on the cell culture supernatant after concentration five-fold after incubation of the detector cell cultures for two weeks. This again shows the strong inhibitory effect of peptides 2, 4, 5, 9, 28 and 29 on HIV-1 synthesis. Inhibition of HIV-2 synthesis by peptides 4, 5, 28 and 29 is also very clearly evident. Nevertheless, in this case the added peptides appear to have a weaker inhibitory effect. Thus, there is no inhibition with peptides 2 and 9, while complete inhibition of virus synthesis is possible with 28 only at the 200 .mu.g/ml dose. Cytotoxicity was found only for peptide No. 9 among all the 41 investigated peptides.
The invention accordingly relates to peptides which contain at least one of the two peptide sequences NPGLLETSEGCRO and AATLEEMMTA and are not larger than 50 amino acids, in particular not larger than 40 amino acids, especially not larger than 30 amino acids, preferably not larger than 20 amino acids. Particularly preferred peptides contain peptides 4, 5, 28 or 29.
Another embodiment of the invention relates to peptides which contain at least one of the two peptide sequences NPGLLETSEGCRO and AATLEEMMTA, where the sequences can be truncated by up to 7 amino acids, preferably by up to 4 amino acids, at the N and/or at the C terminus but without the length falling below 6 amino acids. It is also frequently advantageous to extend the peptides according to the invention by one or more amino acids, for example cysteine, in order to achieve linkage of the peptides to one another or to a carrier.
The invention furthermore relates to derivatives of the two peptide sequences NPGLLETSEGCRQ and AATLEEMMTA in which one or more of the following substitutions can be carried out by methods known to the person skilled in the art: asparagine by glutamine, or glutamine by asparagine, proline by hydroxyproline, leucine by isoleucine or by norleucine, glutamic acid by aspartic acid, threonine by serine or serine by threonine, cysteine by serine, arginine by lysine, alanine by glycine and/or methionine by norleucine. Replacement of a natural amino acid by an unnatural amino acid such as, for example, hydroxyproline or norleucine is preferred. This makes it possible to prevent endogenous proteases cleaving the derivative, which generally results in an increase in the half-life. It is also possible for the derivatives to have improved solubility and/or better absorption than the two peptide sequences according to the invention.
The invention further relates to the preparation by protein chemistry or genetic manipulation and to the use of the peptides according to the invention as pharmaceuticals.